In-vivo molecular imaging is a rapidly advancing field, impacting drug development and testing, research into disease processes, and potentially clinical diagnostic imaging. Optical molecular imaging is a method in which an optical contrast substance is introduced to or activated within an animal, and the resultant signal due to the optical contrast substance, (such as light being absorbed or emitted, whether in a UV, visible or infrared range) is measurable using an optical detector such as a camera to provide one or more images.
For example, optical molecular probes are available which can include fluorescent or luminescent dyes, or absorbing substances, and can be used to target and label specific cell types or activate biochemical processes like bioluminescence. Molecular probes can also be generated by cells in animals transgenically or otherwise altered to do so. Optical molecular imaging, as compared to magnetic resonance imaging (MRI), x-ray or positron-emission imaging, benefits from the fact that such fluorescent, luminescent or absorbing substances can be small, biocompatible molecules.
Optical molecular probes are generally of the following three types: 1) injectable (or otherwise introduced), which are designed to accumulate at the location of a particular target, 2) expressed, via transgenic mutation, or 3) transplanted, following labeling in-vitro of particular cell types. Probes can be normally fluorescent, luminescent or absorbing, or can be activatable, i.e., can change their optical properties in response to environment change.
Many different types of fluorescent and luminescent dyes are available for incorporation into molecular probes including organic and nanoparticle based dyes. Fluorescent dyes require excitation at an appropriate wavelength range for light emission, with the emitted light occurring in a different wavelength range than the excitation range. Luminescent dyes do not require excitation. Recent efforts have focused on the development of dyes which excite and/or emit in the near-infrared (NIR) region, where scattering and absorption in tissue is significantly less, enabling improved penetration and resolution.
In-vivo optical molecular imaging is typically performed on small animals to study the physiologic, pathologic or pharmacologic effects of various drugs or diseases. Molecular imaging can also be performed on humans, and it is hoped that molecular imaging will eventually provide substantial advances in diagnostic imaging. The benefits of in-vivo imaging of small animals are significant because it allows processes and responses to be visualized in real-time in their native environments, and allows longitudinal studies to be performed using the same small animal over time, allowing evaluation of disease progression or response to treatment. Further, in-vivo imaging of small animals reduces the number of animals required for a study, and can reduce the variance in studies where disease manifestation varies from animal to animal, such as cancers in-situ.
However, in-vivo optical molecular imaging presents many challenges. The main challenge is overcoming the effects of light scatter and absorption. For example, when a fluorescent or luminescent dye is used to label specific cells, because the cells can be located deep within the body, the light emitted from them will undergo optical scattering because of intervening matter, which is more problematic the deeper the labeled cells are within the body. Thus, unless the labeled cells are located near the outer surface of the animal, the scatter and absorption of light distorts and attenuates the signals emanating therefrom such that the localization, quantification, and host organ identification of the signals can be very difficult. These effects can distort the apparent shape and location of these targeted labeled cells.
One approach to overcome the problems associated with light scattering involves the use of subcutaneous xenografts (or transplanted cells or tissue) whose superficial location simplifies the localization of labeled cells. However, for transplanted cancer cells, xenografts often do not resemble the human disease, as these cells are often surrounded by a pseudo-capsule, have limited chances to invade major anatomical structures, and rarely spread metastasis. Thus, the study of orthotopic disease models, i.e., cells in their native location, is generally preferred.
Another problem associated with the use of fluorescent and luminescent molecular probes is that these are generally designed such that measurable light signals emanate from the labeled cells, with little or no detectable signals emanating from adjacent tissue or organs, so that the localization of the labeled cells is often uncertain. In the absence of a non-invasive tool for verifying the anatomical location thereof, scientists are limited to ex-vivo histological examination of the labeled cells. This can significantly increase study cost and time, and can degrade data owing to inter-animal variability.
Further, auto-fluorescence and non-specific labeling within the body confounds attempts to identify the true location of labeled cells. Auto-fluorescence arises from intrinsic fluorophores such as tryptophan, collagen, NADH and porphyrins, and also from chemicals in many common animal foods, causing marked fluorescence in the intestines. Multispectral optical imaging has been used to improve image contrast and isolate signals from the labeled cells in the presence of auto-fluorescence. These multispectral imaging systems are typically implemented using a plurality of optical fixed filters or alternatively tunable filters positioned in front of an optical detector to allow light within a predetermined wavelength range to be recorded, and to record a series of images at different wavelength ranges. Other means of multispectral imaging including dispersive and snapshot systems are also applicable. The resulting image sets are equivalent to each image pixel having its own emission spectrum. Analysis can be performed to separate pixels with differing spectral signatures and hence different constituent fluorophores. For example, this approach has been implemented by Cambridge Research Instruments (CRI Maestro™ Woburn Mass.) using liquid crystal tunable filters positioned in front of a CCD camera.
Various three dimensional (3D) imaging systems have also been developed to overcome some of these difficulties, including for example Xenogen's IVIS Imaging System 3D series, which uses optical tomography for imaging small animals such as mice. Such systems rely on making many measurements between light sources and detectors where the relative positions are varied to create a tomographic data set. To create 3D images, an image reconstruction algorithm is required which utilizes a simulation of the likely scattered propagation of light through the tissue based on its geometry and estimated background optical properties. The motivations for creating 3D images of the distribution of a molecular probe within the animal include the desire for 3D localization of the contrast in the animal to aid in interpretation of its anatomical position. However, such systems are highly complex, expensive, and still suffer from a lack of landmark anatomical structures to allow the actual anatomical location of the labeled cells to be identified. While the IVIS Imaging System offers a generic computerized 3D mouse anatomical atlas to overlay with acquired image data to aid in organ identification, matching generic anatomy to each individual mouse is not always accurate and is highly sensitive to mouse age, size, repositioning, and any distortions within the optical images.
Other multi-modality imaging systems have also been developed which combine optical imaging with other imaging modalities such as x-ray, micro-CT, magnetic resonance imaging (MRI), or ultrasound. Such systems significantly increase the cost and complexity of the imaging process, especially when simultaneous image acquisition is attempted. In addition, x-ray contrast of bone and the non-specific contrast of ultrasound will not provide good delineation of the internal soft tissue organs. Since the imaging radiation and geometries are also different between modalities, co-registration with the optical images is problematic.
Various known approaches to optical molecular imaging are described below, with these descriptions also including descriptions of various optical contrast substances which can be optically imaged. These descriptions are not inclusive of all possible embodiments of this invention. In one approach, it is possible to conjugate optical dyes to active molecules such as peptides and antibodies that will specifically bind to targeted cell types. These targeted fluorophore probes are then injected, usually intravenously, and localize at the site of the target. For example, under-glycosylated mucin-1 antigen (uMUC-1) is overexpressed in greater than 90% of human breast, ovarian, pancreatic, colorectal, lung, prostate, colon and gastric carcinomas. A uMUC-1 targeting molecular probe has been developed which carries two fluorescent molecules (FITC and Cy5.5) in addition to a cross-linked iron oxide (CLIO) particle which provides magnetic resonance imaging (MRI) contrast. Mice can be subcutaneously implanted with both uMUC-1 positive and uMUC-1 negative tumors, and then injected with the uMUC-1 targeted probe approximately 24 hours prior to MRI and then optical imaging with a commercially available two dimensional imaging system. Both imaging modalities show good localization of the probe to the uMUC-1 positive tumors.
Labeled cell transplantation is a method that includes labeling specific live cells in-vitro and then transplanting them in-vivo, and provides a way to study cell migration, such as the circulation of tumor cells in the blood stream, or for investigating responses of cells in their native environments to pharmacology or interventions. In this approach, the need for target specificity is reduced. This method is feasible for longitudinal imaging of the migration of cy5.5 (optical)+CLIO (MRI) labeled, transplanted human pancreatic islet cells in-vivo. Islet transplantation into the liver is a promising human treatment for diabetes. By transplanting labeled cells into the kidney capsule or liver of mice, their migration and viability can be longitudinally monitored in-vivo.
Activatable fluorescence is the use of fluorescent molecular probes that become fluorescent in the presence of a particular molecule such as enzyme. For example, to evaluate the role of protease cathepsin-B expression in tumor invasion of 9L gliosarcomas, a cathepsin-B sensitive fluorescent probe can be injected intravenously a couple of weeks after implantation of such tumors into the brains of mice. Approximately sixteen hours after probe injection, sufficient fluorescence can be detected to visualize the tumors non-invasively in-vivo and confirming cathepsin-B expression.
Transgenic bioluminescence is a method wherein developed transgenic mice have localized expression of a bioluminescent enzyme, commonly luciferase. For prostate imaging, a prostate-specific antigen (PSA) promoter can be targeted to express luciferase, and a D-luciferin can be injected to react with luciferase to create bioluminescence at the site of luciferase expression. It is typical to wait approximately 25 minutes for this process to be complete before imaging, and to subtract background luminescence.
Transgenic fluorescence involves the use of varieties of green, yellow and red fluorescent proteins (GFP, YFP and RFP respectively for example) in transgenic animals. Specific cells can be targeted to express a fluorescent protein in normal or diseased animals. Transgenic expression of fluorescent molecules can be present from birth in genetically modified animals, but can also be achieved locally in-vivo using fluorescent protein-carrying adenoviruses. It is also possible to transplant transgenic tissues/cells into normal animals, where they continue to express their fluorescent proteins. In a recent study, mice expressing GFP driven by nestin, a stem cell marker expressed in nascent blood vessels, received orthotopic transplantation of RFP expressing human pancreatic tumor cells. An angiogenic inhibitor was given to a subset of the mice over 14 days until they were sacrificed. Ex-vivo microscopy of both the RFP and GFP allowed vessel growth and tumor growth to be evaluated independently.